Srsf3-mediated alternative RNA splicing in the facial mesenchyme

Srsf3介导的面部间质中的替代RNA剪接

• 批准号: 10190759
• 负责人: Brenna Jo Clay Dennison
• 金额: $ 1.8万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10190759
• 关键词: 1-Phosphatidylinositol 3-Kinase; Ablation; Address; Alleles; Alternative Splicing; Antibodies; Arginine; Binding; Binding Sites; Biochemical; Biological Process; Biology; Cell Lineage; Cell membrane; Cells; Cellular biology; Cephalic; Cleft Lip; Cleft Palate; Congenital Abnormality; Craniofacial Abnormalities; Defect; Development; Dilated Cardiomyopathy; Disease; Embryo; Embryonic Development; Event; Exhibits; Face; Genes; Genetic; Genetic Epistasis; Goals; Human; Immunoprecipitation; Incidence; Individual; Knock-in; Length; Ligands; Live Birth; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Maxilla; Maxillary Prominence; Mediating; Mesenchyme; Messenger RNA; Molecular; Morphogenesis; Morphology; Mus; Muscular Dystrophies; Mutation; Nasal cavity; Neural Crest Cell; Ontology; Oral; PDGFRA gene; Phenotype; Phosphorylation; Phosphotransferases; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor alpha Receptor; Play; Post-Translational Protein Processing; Process; Protein Array; Proteins; RNA Binding; RNA Processing; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Retinitis Pigmentosa; Role; Serine; Signal Transduction; Specificity; Techniques; Time; Tissues; Transcript; Transfection; United States; Validation; Xenopus; base; cell motility; cleft lip and palate; cohesion; craniofacial; craniofacial development; crosslink; experimental study; in vivo; insight; mouse model; mutant; mutant mouse model; nervous system disorder; novel; novel therapeutics; palatal shelves; palatogenesis; phosphoproteomics; programs; protein function; recruit; transcriptome sequencing

Project Summary Craniofacial development is a critical morphological event during embryogenesis, defects in which result in highly prevalent human birth defects such as cleft lip and palate (CL/P). In both humans and mice this process relies on signaling through the platelet-derived growth factor receptor alpha (PDGFRa). Mutations in human PDGFRA are associated with CL/P and mouse models with mutations in this gene similarly display facial clefting phenotypes. Identification of phosphorylation targets of PI3K/Akt downstream of PDGFRa signaling in E13.5 primary mouse embryonic palatal mesenchyme cells revealed an enrichment for proteins that regulate RNA splicing, including the RNA-binding protein (RBP) Srsf3. While dysregulation of alternative RNA splicing has been shown to cause numerous diseases in humans, the role of RBPs in regulating alternative splicing in neural crest cells (NCCs) and their derivatives in the facial mesenchyme has been understudied. The aim of this proposal is to examine the tissue-specific alternative RNA splicing mediated by Srsf3 in the facial mesenchyme and the effect of PI3K/Akt-mediated PDGFRa signaling on Srsf3 activity during midface development. First, the role of Srsf3 during craniofacial development in vivo will be characterized using a conditional Srsf3fl allele in combination with the NCC-specific Wnt1-CreTg driver. The timing and extent of NCC migration will be analyzed in this setting as well as the expression of markers specific to individual facial processes. Second, an RNA- sequencing experiment will be performed to identify transcripts that are differentially alternatively spliced between E11.5 Srsf3fl/fl;Wnt1-Cre+/+ and Srsf3fl/fl;Wnt1-Cre+/Tg maxillary processes. Validation of the differential alternative splicing of select transcripts will be performed using semi-quantitative PCR. Finally, the effect of Akt phosphorylation on Srsf3 subcellular localization, RNA binding and sequence specificity will be assessed through the transfection of Srsf3 wild-type and phosphomutant GFP fusion constructs and enhanced crosslinking and immunoprecipitation followed by sequencing, respectively, in the presence or absence of PDGF-AA ligand. Further, genetic epistasis experiments will be performed between PdgfraPI3K and Srsf3fl mice and the resulting craniofacial phenotypes analyzed to determine if the PdgfraPI3K/PI3K palatal clefting defect is rescued or exacerbated upon loss of Srsf3 in the NCC lineage. This project will investigate a potentially novel role for Srsf3 as a splicing regulator in NCCs and their derivatives and explore how post-translational modification of this protein downstream of PDGFRa signaling contributes to midface development. These studies will provide significant insight into the mechanisms underlying mammalian craniofacial morphogenesis and new therapeutic directions for the treatment of human craniofacial birth defects such as CL/P.

项目摘要 颅面发育是胚胎发生过程中的一个重要的形态事件，其结果是畸形 在非常普遍的人类出生缺陷中，如唇腭裂(CL/P)。在人类和小鼠身上，这一过程 依赖于通过血小板衍生生长因子受体α(PDGFRA)的信号。人类的基因突变 PDGFRA与CL/P相关，该基因突变的小鼠模型类似地表现为面部裂隙 表型。E13.5中PDGFRA信号下游PI3K/Akt磷酸化靶点的鉴定 原代小鼠胚胎腭部间充质细胞富含调节RNA的蛋白质 剪接，包括RNA结合蛋白(RBP)Srsf3。而另一种RNA剪接的失调 已被证明在人类中引起多种疾病，限制性商业惯例在调节神经选择性剪接中的作用 面部间充质中的冠状细胞(NCC)及其衍生物的研究还不够深入。这样做的目的是 建议研究面部间充质中由srsf3介导的组织特异性替代RNA剪接。 以及PI3K/Akt介导的PDGFRA信号对面中部发育过程中Srsf3活性的影响。首先， 在活体颅面发育过程中，Srsf3的作用将使用一个条件的Srsf3f1等位基因来表征。 与NCC特定的WNT1-CreTg驱动程序相结合。将分析NCC迁移的时间和范围 在这种情况下，以及特定于个别面部过程的标记的表达。第二，一种RNA- 将进行测序实验，以识别差异或选择性剪接的转录本 在E11.5Srsf3fl/fl；Wnt1-Cre+/+和Srsf3fl/fl之间；Wnt1-Cre+/Tg上颌突之间。对差异的验证 选择转录本的选择性剪接将使用半定量聚合酶链式反应进行。最后，Akt的效果 将通过以下方法评估Srsf3亚细胞定位、RNA结合和序列特异性的磷酸化 Srsf3野生型和突变型GFP融合表达载体的构建及增强交联剂的研究 在存在或不存在PDGF-AA配体的情况下，分别进行免疫沉淀和测序。 此外，将在PdgfraPI3K和Srsf3f1小鼠之间进行遗传上位性实验，并由此产生 分析颅面表型以确定PdgfraPI3K/PI3K腭裂畸形是挽救还是 在NCC血统中失去Srsf3后，情况进一步恶化。该项目将调查srsf3潜在的新角色 作为NCC及其衍生物中的剪接调节因子，并探索该基因的翻译后修饰 PDGFRA信号的下游蛋白参与了面中部的发育。这些研究将提供 哺乳动物颅面形态发生机制的重大洞察和新疗法 CL/P等人类头面部出生缺陷的治疗方向